I. Field of the Invention
The present invention relates generally to the field of cellular biology and imaging, and more specifically relates to new zinc sensing compounds and their use in microscopy, cytometry and spectroscopy for the detection of zinc ion release by cells in normal and pathologic states.
II. Description of Related Art
A variety of mammalian cells, including the pancreatic beta cells (Falkmer and Pihl, 1968; Kristiansen et al., 2001), submandibular salivary gland (Frederickson et al., 1987), the prostate epithelial cells (Sorensen et al., 1997), paneth cells in the crypts of Lieberkühn (Giblin et al., 2006; Muller and Geyer, 1969), mast cells (Gustafson, 1967; Ho et al., 2004), granulocytes (Goldberg et al., 1993; Ieshchenko et al., 1994), pituitary cells (Thorlacius-Ussing, 1987), and certain neurons of the central nervous systems (Birinyi et al., 2001; Haug, 1967), etc., contain zinc ion (Zn2+) in their secretory granules. Upon stimulation, these cells release the contents of their secretory granules into extracellular medium, during which Zn2+ is co-released (Frederickson et al., 2005).
FluoZin-3 is the first fluorescent Zn2+ sensor reported for monitoring Zn2+/insulin release in cultured beta cells (Gee et al., 2002). Since FluoZin-3 is applied to the extracellular bath, the sensitivity of detecting local Zn2+ release near the plasma membrane is compromised by the background fluorescence from the bulk solution. Consequently FluoZin-3 imaging has been largely limited to the use of total internal reflection of fluorescence (TIRF) microscopy in order to study secretion at the interface between a cell and the underlying glass coverslip (Michael et al., 2006). Also, FluoZin-3 binds to Zn2+ with nanomolar affinity (Zn2+ dissociation constant Kd(Zn2+)=15 nM), but it also binds Ca2+ with a Kd(Ca2+) of about 10 μM. Since extracellular Ca2+ concentration is higher than 1 mM, Ca2+ effectively competes Zn2+ for FluoZin-3 binding, thus limiting the capacity of FluoZin-3 to sense Zn2+. Further, since FluoZin-3 also binds to other heavy metal ions with high affinity, assaying buffers have to be prepared from the highest grade of salts available and treated with the Chelex resin to remove contaminating heavy metals (Qian et al., 2003). These limitations lower the sensitivity of Zn2+ detection, complicate and lengthen the assaying process, and discourage the routine application of the assay.
Another reported imaging assay of insulin release relies on transfecting cells with GFP tagged insulin or C-peptide. When these fluorescent fusion proteins are expressed in cells, they are incorporated into insulin secretory granules. Exocytosis of these labeled proteins can be captured by total internal reflection fluorescence (TIRF) microscopy (Michael et al., 2007; Nagamatsu, 2006; Ohara-Imaizumi et al., 2002). Since TIRF imaging only detects fluorescence signal at the cell membranes that directly contact a glass coverslip, this method cannot provide information about secretion in 3 dimensions, thus preventing studying exocytosis of cells in the organotypic culture or in the intact islet where normal cell-cell contact is maintained. Further, since the assay relies on transfection and expression of fusion proteins which typically takes 24-48 hours, primary beta cells (including primary human beta cells) may alter their secretory behavior during this time in culture.
In two photon extracellular polar tracer imaging (TEP) (Kasai et al., 2005), islets are immersed in a solution containing a polar, membrane impermeant tracer such as sulforhodamine-B (SRB). Since the assay was based on changes in membrane morphology (forming Ω shaped membrane profile) during secretion, its fluorescence readout is susceptible to artifacts caused by motion or by membrane extension, retraction or ruffling that is unrelated to exocytosis. Further, the assay only detects the dye filling of secretory granules, which does not necessarily correlate with the insulin release activity.
Thus, although a number of techniques exist to detect Zn2+ release in intact cells, each of these has distinct limitations and disadvantages. New and improved compositions and methods for cellular imaging of zinc ion release are therefore needed.